Virginia A. Lightner

Hexabrachion Protein (Tenascin, Cytotactin, Brachionectin) in Connective Tissues, Embryonic Brain, and Tumors

Publisher Summary The hexabrachion is a large oligomeric glycoprotein of the extracellular matrix (ECM). It is synthesized at very specific times and locations during embryonic development, it is absent or restricted in most adult tissues, and it is prominently expressed in a variety of tumors. The protein is present in some apparently stable locations, but is especially prominent in states of development and growth, both embryonic and cancerous. The hexabrachion protein has been discovered independently by several laboratories, each discovery showing a different context of its biology, and each laboratory giving it a different name. The various names given to the protein are listed in the chapter in tabulated form. The survey of tissues showing high concentrations of tenascin provides intriguing clues about its possible functions. Roles in stimulating cell growth, in cell migration, and in cell adhesion have been postulated for some of these locations. Other tissues suggest that tenascin has little direct interaction with most cells, but may play a role in structuring the fibers of the ECM.

Tenascin expression in human glioma cell lines and normal tissues

Tenascin expression was evaluated in 21 human glioma cell lines and in normal adult tissue extracts by Western and Northern blotting. The cell lines differed in their relative expression of tenascin in the cell-associated and supernatant compartments. Glioma cell line tenascin production was not uniformly stimulated by changes in fetal bovine serum concentration in the growth media. In most glioma cell lines and normal tissue extracts, reducing Western blots and Northern blots revealed two tenascin species, respectively: a major 340 kDa polypeptide and a 9 kb RNA transcript accompanied by a less intense 250 kDa polypeptide and 7 kDa RNA species. In U-87 MG and in normal adult kidney extracts, however, the 250 kDa band and 7 kb transcript were more prominent. Quantitation of tenascin in the glioma lines revealed variable levels that were significantly higher than those in the tissue extracts.

Tenascin expression in prostatic hyperplasia, intraepithelial neoplasia, and carcinoma

The expression of tenascin, an extracellular matrix glycoprotein, was studied in three human prostatic carcinoma cell lines by Northern and Western blot analyses and in human prostate tissues by immunohistochemistry and Western blot analysis. All three carcinoma cell lines expressed tenascin mRNA and protein, which were found predominantly in secreted form in culture supernatant. By immunohistochemistry, fetal prostatic tissue showed strong and diffuse tenascin immunoreactivity around developing glands. Normal adult prostatic tissue revealed only focal, scant periglandular and stromal immunoreactivity around acini and ducts. Most cases of hyperplasia and intraepithelial neoplasia showed variable periglandular immunostaining. Tenascin periglandular staining with diffuse stromal extension was noted with all grades of adenocarcinoma; however, the intensity was variable and appeared unrelated to the histologic grade. Metastatic prostatic carcinoma showed strong immunoreactivity in lymph nodes and bone marrow samples, with only weak reactivity of the normal connective tissue framework in both tissues. Western blot analysis of prostatic hyperplasia and carcinoma demonstrated the large and small isoforms of tenascin. These findings suggest a prominent role for tenascin in stromal alterations associated with both benign and malignant prostatic epithelial growth processes.

Purification of hexabrachion (tenascin) from cell culture conditioned medium, and separation from a cell adhesion factor.

We describe a protocol for purifying hexabrachion from conditioned medium of cell cultures, using gel filtration chromatography on Sephacryl 500, followed by anion-exchange chromatography on a Mono Q column, followed optionally by a second gel filtration or zone sedimentation on glycerol gradients. The protocol has several advantages over previous procedures based on affinity chromatography on monoclonal antibodies. Perhaps foremost, the protein is never exposed to the denaturing solvents that are required for elution from the antibody column. The Mono Q column also separated hexabrachion from a prominent cell adhesion activity that eluted with the hexabrachion on the first gel filtration, and co-sedimented with hexabrachions on glycerol gradients. The cell adhesion fractions showed several bands between 190 and 400 kDa. A single band at 220 kDa stained prominently with a polyclonal antibody against mouse EHS laminin, and a band at 190 kDa stained with a monoclonal antibody against s-laminin. The purification protocol gave hexabrachion at high concentration and with no detectable contamination by fibronectin or laminin. The highest yield of hexabrachion (1-4 mg from 400 ml of conditioned medium) was from human glioblastoma cell cultures, but the same procedure allowed us to purify and characterize the rat hexabrachion. Protein purified from primary cultures of rat embryo fibroblasts showed approximately equal amounts of three subunit sizes: 280, 230, and 220 kDa. These different subunits, presumably derived from alternative RNA splicing, appeared to be segregated into large and small hexabrachions, which could be separated on glycerol gradients.

Divalent cation-dependent adhesion at the myotendinous junction: ultrastructure and mechanics of failure.

SummaryJunctional microfibrils, which span the lamina lucida of the vertebrate myotendinous junction, are thought to function in force transmission at the junction. This hypothesis has been tested by disrupting junctional microfibrils through elimination of extracellular divalent cations, and determining the effects of this treatment on the ultrastructure and mechanics of whole frog skeletal muscles passively stretched to failure. Muscles incubated in divalent cation-free solution failed exclusively in the lamina lucida of the myotendinous junction, while control muscles all failed within the muscle fibres, several millimetres away from the junction. Failure sites from divalent cation-free muscles incubated with antibodies against collagen type IV, laminin, and tenascin showed no labelling of the avulsed ends of the muscle fibres, indicating that remnants of junctional microfibrils observed on the cell surface are not composed of any of these extracellular proteins. All three proteins were present on the tendon side of the failure site, confirming that the lamina densa remains attached to the tendon. Breaking stress for control muscles was 3.47×105 N m-2, and for divalent cation-free muscles, 1.84×105 N m-2, or approximately half the control value. Breaking strain averaged 1.17 for divalent cation-free muscles and 1.39 for controls, although the difference was not significant. We conclude that junctional microfibrils are components of a divalent cation-dependent adhesion mechanism at the myotendinous junction. In addition, ultrastructural analysis of divalent cation-free fibres stretched just short of failure suggests that a second, divalent cation-independent mechanism persists along the non-junctional cell surface, and can transmit substantial passive tension from myofibrils laterally to the extracellular matrix, bypassing the failed myotendinous junction.

Localization of Tenascin in Uterine Sarcomas and Partially Transformed Endometrial Stromal Cells

Normal mesenchymal cells within developing embryonic organs and transformed stromal cells in organs undergoing spontaneous carcinogenesis have the capacity for normal or altered expression of the extracellular matrix glycoprotein tenascin (Tn). Mesenchymal cell constituents of normal adult organs show only a very limited tendency to deposit Tn in their extracellular matrix. In the present study, we investigated whether malignant human mesenchymal cells derived from uterine sarcomas or normal human endometrial stromal cells partially transformed via transfection with selected oncogenes have the capacity to produce and deposit Tn. We reached the following conclusions: (1) compared with normal endometrial tissues, uterine sarcomas show heterogeneous, but increased, immunoreactive staining patterns exclusively within the extracellular compartment, regardless of the histologic subtype of the tumor; (2) in vitro, all normal and transfected stromal cells and cell lines examined secreted Tn into the tissue culture medium; (3) this secretory ability was not translated into morphologic uniformity, since immunoreactivity detected by confocal laser scanning microscopy was observed in only selected cell populations; (4) also, the deposition and the incorporation of Tn depended upon the density of transfected cells, and (5) double-staining experiments revealed that Tn could always be localized in close proximity to fibronectin. In summary, the production of Tn is increased in most cases of human uterine sarcoma. The capacity of stromal cells to deposit Tn in a matrix-like structure in vitro, rather than increase production of Tn, is correlated with the degree of neoplastic progression.

Monoclonal Antibody 127 Recognizes a Subpopulation of Chicken Hexabrachion/Tenascin

Hexabrachion/tenascin is an extracellular matrix protein with a distinct spatial and temporal distribution in tissue (Erickson and Lightner, 1988). In chicken embryo fibroblast cultures it is isolated as a hexameric protein with disulfide linked subunits of 250, 225 and 215 Kd (Chiquet and Fambrough, 1984a,b; Erickson and Lightner, 1988). These subunits have very similar proteolytic cleavage patterns, indicating they are related peptides (Chiquet and Fambrough, 1984b). Pulse chase experiments (Chiquet and Fambrough, 1984b) and recently published sequence data (Jones et al.,1988; Pearson et al.,1988) suggest they result from alternative splicing of the tenascin mRNA. However, nothing is known about the functional differences between these subunits or how they are assembled into the hexameric structure.